University of Melbourne Department of Paediatrics, Parkville, Victoria, Australia.
Nat Protoc. 2011 Mar;6(3):388-404. doi: 10.1038/nprot.2010.179. Epub 2011 Mar 3.
Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.
软骨蛋白聚糖聚集素的蛋白水解是关节炎的一个特征。我们提出了一种基于为大型动物物种开发的传统方法，分析 3 周大的小鼠股骨头软骨体外培养中聚集素水解的方法。研究人员可以选择仅检测释放到培养基中的几种聚集素片段的简单分析，也可以选择检测存在于培养基和软骨基质中的所有片段的更全面的研究。该方案包括 (i) 软骨培养和可选的软骨提取，(ii) 用于定量聚集素的快速简单比色测定，以及 (iii) 识别分配到培养基或软骨隔室的特定聚集素片段的新表位 Western 印迹。小鼠和大型动物方法之间的关键区别在于，给定样品中聚集素的比例是相对于总聚集素而不是相对于组织湿重进行归一化的。这种必要的与传统的背离源于这样一个事实，即粘在小鼠软骨上的微小液体体积可以增加组织湿重，从而导致不可接受的误差。该方案广泛适用于转基因小鼠的体外分析，特别是那些影响软骨重塑、关节炎疾病和骨骼发育的突变小鼠。该方案稳健可靠，完成需要 7-11 天。
